Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology, antibiotic resistance and virulence / El sistema Tol-Pal de Acinetobacter baumannii es importante para la morfología celular, la resistencia a los antibióticos y la virulencia

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most successful human pathogens worldwide and responsible for hospital-acquired infections. This is due to its outstanding potential to adapt to very different environments, to persist in the human host and most important, its ability to develop multidrug resistance. Our combined approach of genomic and phenotypic analyses led to the identification of the envelope spanning Tol-Pal system in A. baumannii. We found that the deletion of the tolQ, tolR, tolA, tolB, and pal genes affects cell morphology and increases antibiotic sensitivity, such as the ∆tol-pal mutant exhibits a significantly increased gentamicin and bacitracin sensitivity. Furthermore, Galleria mellonella caterpillar killing assays revealed that the ∆tol-pal mutant exhibits a decreased killing phenotype. Taken together, our findings suggest that the Tol-Pal system is important for cell morphology, antibiotic resistance, and virulence of A. baumannii.(AU)

Intervenções psicológicas necessárias: a prática como residente no serviço de medicina fetal / Necessary psychological interventions: the practice as a resident in the fetal medicine service / Intervenciones psicológicas necesarias: la práctica como residente en el servicio de medicina fetal

Com os avanços tecnológicos e o aprimoramento da prática médica via ultrassonografia, já é possível detectar possíveis problemas no feto desde a gestação. O objetivo deste estudo foi analisar a prática do psicólogo no contexto de gestações que envolvem riscos fetais. Trata-se de um estudo qualitativo sob formato de relato de experiência como psicólogo residente no Serviço de Medicina Fetal da Maternidade Escola da Universidade Federal do Rio de Janeiro (UFRJ). Os registros, feitos por observação participante e diário de campo, foram analisados em dois eixos temáticos: 1) intervenções psicológicas no trabalho em equipe em consulta de pré-natal, exame de ultrassonografia e procedimento de amniocentese; e 2) intervenções psicológicas em casos de bebês incompatíveis com a vida. Os resultados indicaram que o psicólogo nesse serviço é essencial para atuar de forma multiprofissional na assistência pré-natal para gravidezes de alto risco fetal. Ademais, a preceptoria do residente é relevante para sua formação e treinamento para atuação profissional no campo da psicologia perinatal.(AU)

Face to the technological advances and the improvement of medical practice via ultrasound, it is already possible to detect possible problems in the fetus since pregnancy. The objective of this study was to analyze the psychologist's practice in the context of pregnancies which involve fetal risks. It is a qualitative study based on an experience report as a psychologist trainee at the Fetal Medicine Service of the Maternity School of UFRJ. The records, based on the participant observation and field diary, were analyzed in two thematic axes: 1) psychological interventions in the teamwork in the prenatal attendance, ultrasound examination and amniocentesis procedure; and 2) psychological interventions in cases of babies incompatible to the life. The results indicated that the psychologist in this service is essential to work in a multidisciplinary way at the prenatal care for high fetal risk pregnancies. Furthermore, the resident's preceptorship is relevant to their education and training for professional performance in the field of Perinatal Psychology.(AU)

Con los avances tecnológicos y la mejora de la práctica médica a través de la ecografía, ya se puede detectar posibles problemas en el feto desde el embarazo. El objetivo de este estudio fue analizar la práctica del psicólogo en el contexto de embarazos de riesgos fetal. Es un estudio cualitativo basado en un relato de experiencia como residente de psicología en el Servicio de Medicina Fetal de la Escuela de Maternidad de la Universidade Federal do Rio de Janeiro (UFRJ). Los registros, realizados en la observación participante y el diario de campo, se analizaron en dos ejes temáticos: 1) intervenciones psicológicas en el trabajo en equipo, en la consulta prenatal, ecografía y los procedimientos de amniocentesis; y 2) intervenciones psicológicas en casos de bebés incompatibles con la vida. Los resultados señalaron como fundamental la presencia del psicólogo en este servicio trabajando de forma multidisciplinar en la atención prenatal en el contexto de embarazos de alto riesgo fetal. Además, la tutela del residente es relevante para su educación y formación para el desempeño profesional en el campo de la Psicología Perinatal.(AU)

A study of the antibacterial mechanism of pinocembrin against multidrug-resistant Aeromonas hydrophila / Un estudio del mecanismo antibacteriano de la pinocembrina contra Aeromonas hydrophila resistente a múltiples fármacos

Aeromonas hydrophila is a common pathogen in fish that has caused severe economic losses in aquaculture worldwide. With the emergence of bacterial resistance, it is necessary to develop new drugs to combat bacterial infection, particularly for multidrug-resistant bacteria. In this study, the antibacterial activity of pinocembrin was investigated by observing bacterial growth and microscopic structure, and its mechanism of action was identified by investigating its effect on protein and DNA. The antibacterial susceptibility test indicated that pinocembrin inhibits A. hydrophila growth. The minimal inhibitory concentration and minimum bactericidal concentration were 256 μg/mL and 512 μg/mL, respectively. Ultrastructurally, the bacteria treated with pinocembrin showed surface roughness and plasmolysis. When bacteria were treated with 512 μg/mL pinocembrin, lactate dehydrogenase activity and soluble protein content decreased significantly, and electrical conductivity and DNA exosmosis levels increased by 4.21 ± 0.64% and 15.98 ± 1.93 mg/L, respectively. Staining with 4′, 6-Diamidino-2-phenylindole showed that the nucleic acid fluorescence intensity and density decreased after the treatment with pinocembrin. Pinocembrin may inhibit the growth of A. hydrophila by increasing cell membrane permeability and affecting protein and DNA metabolism. Thus, pinocembrin is a candidate drug for the treatment of A. hydrophila infection in aquaculture.(AU)

Utilização da morfometria celular no microambiente neoplásico e sua relação com a imunoexpressão da interleucina-4 / Use of cellular morphometry in the neoplastic microenvironment and its relationship with the immunoexpression of interleukin-4

A morfometria celular é um tipo de análise quantitativa que utiliza as medidas geométricas para obter informações acerca da morfologia nuclear, citoplasmática e outras medidas gerais das células, sendo que estes parâmetros podem estar alterados devido a processos fisiológicos e patológicos que modificam a morfologia celular normal, sendo uma análise relevante no prognóstico de diversas lesões. Os ceratinócitos possuem morfologia poliédrica com núcleos ovoides, mas podem sofrer alterações com eventos patológicos e fisiológicos, por exemplo, no núcleo. Essas alterações podem estar presentes em neoplasias malignas, como, no carcinoma epidermoide. A interleucina-4 (IL-4) é uma citocina secretada por diversos tipos de células, estando envolvida no desenvolvimento e diferenciação de células Th2, atuando na resposta antiinflamatória e sua expressão parece estar relacionada com o desenvolvimento de algumas neoplasias, incluindo o câncer oral. O objetivo da pesquisa foi realizar análise morfométrica celular (compartimentos nuclear e citoplasmático), identificação de irregularidades nucleares de ceratinócitos malignos e comparar com a imunoexpressão da IL-4 e profundidade de invasão nos casos de Carcinoma Epidermoide de Lábio Inferior (CELI) e Língua Oral (CELO). Foram analisados 30 casos de cada lesão. Para morfometria celular foram analisados 16 ceratinócitos malignos por caso; a imunoexpressão de IL-4 foi analisada no parênquima e estroma das lesões, utilizando softwares de análises de imagens. Foram aplicados os testes estatísticos de Análise de Variância, Kruskal-Wallis e Correlação de Spearman. A imunoexpressão de IL-4 no parênquima foi maior nos casos de CELI em todos os campos analisados (p<0,05); Houve correlação positiva entre área celular total e profundidade de invasão (p=0,038) e negativa entre imunoexpressão da IL-4 no parênquima superficial e perímetro nuclear superficial (p=0,007). Portanto, sugere-se que exista uma relação protetora da imunoexpressão IL-4 com as lesões analisadas, bem como alterações morfométricas dos ceratinócitos malignos com a imunoexpressão da IL-4 nessas lesões (AU).

Cell morphometry is a type of quantitative analysis that uses geometric measurements of cells to obtain information about the nuclear, cytoplasmic and general morphology of cells. Such parameters may be altered due to physiological and pathological processes that modify normal cell morphology, being a relevant analysis in the prognosis of several injuries. Keratinocytes have a polyhedral morphology with ovoid nuclei, but may undergo changes with pathological and physiological events, for example, in the nucleus. These changes may be present in malignant neoplasms, such as squamous cell carcinoma. Interleukin-4 (IL-4) is a cytokine secreted by several types of cells, being involved in the development and differentiation of Th2 cells, acting in the anti-inflammatory response and its expression seems to be related to the development of some neoplasms, including the oral cancer. The objective of the research was to perform cellular morphometric analysis (nuclear and cytoplasmic compartments), identification of nuclear irregularities of malignant keratinocytes and compare with the immunoexpression of IL-4 and depth of invasion in cases of Epidermoid Carcinoma of the Lower Lip (CELI) and Oral Tongue. (CELLO). Thirty cases of each lesion were analyzed. For cell morphometry, 16 malignant keratinocytes were analyzed per case; IL-4 immunoexpression was analyzed in the parenchyma and stroma of the lesions, using image analysis software. The statistical tests of Analysis of Variance, Kruskal-Wallis and Spearman's Correlation were applied. The immunoexpression of IL-4 in the parenchyma was higher in cases of CELI in all analyzed fields (p<0,05); There was a positive correlation between total cell area and depth of invasion (p=0.038) and a negative correlation between IL-4 immunoexpression in the superficial parenchyma and superficial nuclear perimeter (p=0.007). Therefore, it is suggested that there is a protective relationship between IL-4 immunoexpression and the lesions analyzed, as well as morphometric alterations of malignant keratinocytes with IL-4 immunoexpression in these lesions (AU).

A deep hybrid learning pipeline for accurate diagnosis of ovarian cancer based on nuclear morphology.

Nuclear morphological features are potent determining factors for clinical diagnostic approaches adopted by pathologists to analyze the malignant potential of cancer cells. Considering the structural alteration of the nucleus in cancer cells, various groups have developed machine learning techniques based on variation in nuclear morphometric information like nuclear shape, size, nucleus-cytoplasm ratio and various non-parametric methods like deep learning have also been tested for analyzing immunohistochemistry images of tissue samples for diagnosing various cancers. We aim to correlate the morphometric features of the nucleus along with the distribution of nuclear lamin proteins with classical machine learning to differentiate between normal and ovarian cancer tissues. It has already been elucidated that in ovarian cancer, the extent of alteration in nuclear shape and morphology can modulate genetic changes and thus can be utilized to predict the outcome of low to a high form of serous carcinoma. In this work, we have performed exhaustive imaging of ovarian cancer versus normal tissue and developed a dual pipeline architecture that combines the matrices of morphometric parameters with deep learning techniques of auto feature extraction from pre-processed images. This novel Deep Hybrid Learning model, though derived from classical machine learning algorithms and standard CNN, showed a training and validation AUC score of 0.99 whereas the test AUC score turned out to be 1.00. The improved feature engineering enabled us to differentiate between cancerous and non-cancerous samples successfully from this pilot study.

Two nondimensional parameters for characterizing the nuclear morphology.

Nuclear morphology is an important indicator of cell function. It is regulated by a variety of factors such as the osmotic pressure difference between the nucleoplasm and cytoplasm, cytoskeletal forces, elasticity of the nuclear envelope and chromosomes. Nucleus shape and size are typically quantified using multiple geometrical quantities that are not necessarily independent of one another. This interdependence makes it difficult to decipher the implications of changes in nuclear morphology. We resolved this by analyzing nucleus shapes of populations for multiple cell lines using a mechanics-based model. We deduced two independent nondimensional parameters, namely, flatness index and isometric scale factor. We show that nuclei in a cell population have similar flatness but variable scale factor. Furthermore, nuclei of different cell lines segregate according to flatness. Cellular perturbations using biochemical and biomechanical techniques suggest that the flatness index correlates with actin tension and the scale factor anticorrelates with elastic modulus of nuclear envelope. We argue that nuclear morphology measures such as volume, projected area, height etc., are subsumed by flatness and scale factor, which can unambiguously characterize nuclear morphology.

Nuclear Dynamics and Chromatin Structure: Implications for Pancreatic Cancer.

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the 'gold standard' for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology.

Polyphenols Extracted from Shanxi-Aged Vinegar Inhibit Inflammation in LPS-Induced RAW264.7 Macrophages and ICR Mice via the Suppression of MAPK/NF-κB Pathway Activation.

Shanxi-aged vinegar, a traditional Chinese grain-fermented food that is rich in polyphenols, has been shown to have therapeutic effects on a variety of diseases. However, there has been no comprehensive evaluation of the anti-inflammatory activity of polyphenols extracted from Shanxi-aged vinegar (SAVEP) to date. The anti-inflammatory activities of SAVEP, both in RAW 264.7 macrophages and mice, were extensively investigated for the potential application of SAVEP as a novel anti-inflammatory agent. In order to confirm the notion that polyphenols could improve inflammatory symptoms, SAVEP was firstly detected by gas chromatography mass spectrometry (GC-MS). In total, 19 polyphenols were detected, including 12 phenolic acids. The study further investigated the protective effect of SAVEP on lipopolysaccharide-induced inflammation in RAW264.7 macrophages and ICR mice. The results showed that compared with those of the model group, SAVEP could remarkably recover the inflammation of macrophage RAW264.7 and ICR mice. SAVEP can normalise the expression of related proteins via the suppression of MAPK/NF-κB pathway activation, inhibiting the expression of iNOS and COX-2 proteins, and consequently the production of inflammatory factors, thus alleviating inflammatory stress. These results suggest that SAVEP may have a potential function against inflammation.

Interleukin-32θ Triggers Cellular Senescence and Reduces Sensitivity to Doxorubicin-Mediated Cytotoxicity in MDA-MB-231 Cells.

The recently discovered interleukin (IL)- 32 isoform IL-32θ exerts anti-metastatic effects in the breast tumor microenvironment. However, the involvement of IL-32θ in breast cancer cell proliferation is not yet fully understood; therefore, the current study aimed to determine how IL-32θ affects cancer cell growth and evaluated the responses of IL-32θ-expressing cells to other cancer therapy. We compared the functions of IL-32θ in triple-negative breast cancer MDA-MB-231 cells that stably express IL-32θ, with MDA-MB-231 cells transfected with a mock vector. Slower growth was observed in cells expressing IL-32θ than in control cells, and changes were noted in nuclear morphology, mitotic division, and nucleolar size between the two groups of cells. Interleukin-32θ significantly reduced the colony-forming ability of MDA-MB-231 cells and induced permanent cell cycle arrest at the G1 phase. Long-term IL-32θ accumulation triggered permanent senescence and chromosomal instability in MDA-MB-231 cells. Genotoxic drug doxorubicin (DR) reduced the viability of MDA-MB-231 cells not expressing IL-32θ more than in cells expressing IL-32θ. Overall, these findings suggest that IL-32θ exerts antiproliferative effects in breast cancer cells and initiates senescence, which may cause DR resistance. Therefore, targeting IL-32θ in combination with DR treatment may not be suitable for treating metastatic breast cancer.

Ongoing repair of migration-coupled DNA damage allows planarian adult stem cells to reach wound sites.

Mechanical stress during cell migration may be a previously unappreciated source of genome instability, but the extent to which this happens in any animal in vivo remains unknown. We consider an in vivo system where the adult stem cells of planarian flatworms are required to migrate to a distal wound site. We observe a relationship between adult stem cell migration and ongoing DNA damage and repair during tissue regeneration. Migrating planarian stem cells undergo changes in nuclear shape and exhibit increased levels of DNA damage. Increased DNA damage levels reduce once stem cells reach the wound site. Stem cells in which DNA damage is induced prior to wounding take longer to initiate migration and migrating stem cell populations are more sensitive to further DNA damage than stationary stem cells. RNAi-mediated knockdown of DNA repair pathway components blocks normal stem cell migration, confirming that active DNA repair pathways are required to allow successful migration to a distal wound site. Together these findings provide evidence that levels of migration-coupled-DNA-damage are significant in adult stem cells and that ongoing migration requires DNA repair mechanisms. Our findings reveal that migration of normal stem cells in vivo represents an unappreciated source of damage, which could be a significant source of mutations in animals during development or during long-term tissue homeostasis.

Histopathological Differentiation of Antemortem and Postmortem Electrical Burn Mark Produced by Low Voltage.

ABSTRACT: The study was undertaken to differentiate antemortem electrical (AME) and postmortem electrical (PME) burn marks with the help of histopathology. The electrical burn mark was produced on 25 dead bodies. Alongside 25 cases of electrocution deaths were included for comparison. Slides were prepared and stained with hematoxylin-eosin stains. Intraepidermal and subepidermal separation; coagulative necrosis of the epidermis; nuclear elongation and hyperchromasia of epidermal cells; homogenization of the dermis; nuclear elongation and hyperchromasia of hair follicles, sweat glands, sebaceous glands, and blood vessel endothelium were studied for histopathological changes and graded. The findings of the study suggest that the histopathological changes in electrical burn marks are due to the physical effect of heat produced by the electric current. The classical histopathological features of electrical burn mark cannot differentiate between AME and PME burn marks. However, careful evaluation of grading of the dermal changes can be helpful in differentiating AME and PME burn marks. Highest grade of dermal thickness homogenization and highest grade of nuclear elongation of dermal appendages were significantly more in the antemortem electrical burn marks than PME burn marks.

Autophagy displays divergent roles during intermittent amino acid starvation and toxic stress-induced senescence in cultured skeletal muscle cells.

Due to the ever-expanding functions attributed to autophagy, there is widespread interest in understanding its contribution to human physiology; however, its specific cellular role as a stress-response mechanism is still poorly defined. To investigate autophagy's role in this regard, we repeatedly subjected cultured mouse myoblasts to two stresses with diverse impacts on autophagic flux: amino acid and serum withdrawal (Hank's balanced salt solution [HBSS]), which robustly induces autophagy, or low-level toxic stress (staurosporine, STS). We found that intermittent STS (int-STS) administration caused cell cycle arrest, development of enlarged and misshapen cells/nuclei, increased senescence-associated heterochromatic foci and senescence-associated ß-galactosidase activity, and prevented myogenic differentiation. These features were not observed in cells intermittently incubated in HBSS (int-HB). While int-STS cells displayed less DNA damage (phosphorylated H2A histone family, member X content) and caspase activity when administered cisplatin, int-HB cells were protected from STS-induced cell death. Interestingly, STS-induced senescence was attenuated in autophagy related 7-deficient cells. Therefore, while repeated nutrient withdrawal did not cause senescence, autophagy was required for senescence caused by toxic stress. These results illustrate the context-dependent effects of different stressors, potentially highlighting autophagy as a distinguishing factor.

Machine-Learning-Based Approach to Decode the Influence of Nanomaterial Properties on Their Interaction with Cells.

In an in vitro nanotoxicity system, cell-nanoparticle (NP) interaction leads to the surface adsorption, uptake, and changes into nuclei/cell phenotype and chemistry, as an indicator of oxidative stress, genotoxicity, and carcinogenicity. Different types of nanomaterials and their chemical composition or "corona" have been widely studied in context with nanotoxicology. However, rare reports are available, which delineate the details of the cell shape index (CSI) and nuclear area factors (NAFs) as a descriptor of the type of nanomaterials. In this paper, we propose a machine-learning-based graph modeling and correlation-establishing approach using tight junction protein ZO-1-mediated alteration in the cell/nuclei phenotype to quantify and propose it as indices of cell-NP interactions. We believe that the phenotypic variation (CSI and NAF) in the epithelial cell is governed by the physicochemical descriptors (e.g., shape, size, zeta potential, concentration, diffusion coefficients, polydispersity, and so on) of the different classes of nanomaterials, which critically determines the intracellular uptake or cell membrane interactions when exposed to the epithelial cells at sub-lethal concentrations. The intrinsic and extrinsic physicochemical properties of the representative nanomaterials (NMs) were measured using optical (dynamic light scattering, NP tracking analysis) methods to create a set of nanodescriptors contributing to cell-NM interactions via phenotype adjustments. We used correlation function as a machine-learning algorithm to successfully predict cell and nuclei shapes and polarity functions as phenotypic markers for five different classes of nanomaterials studied herein this report. The CSI and NAF as nanodescriptors can be used as intuitive cell phenotypic parameters to define the safety of nanomaterials extensively used in consumer products and nanomedicine.

Linalool-Loaded Glutathione-Modified Gold Nanoparticles Conjugated with CALNN Peptide as Apoptosis Inducer and NF-κB Translocation Inhibitor in SKOV-3 Cell Line.

BACKGROUND: Linalool is a monoterpene compound with various potential therapeutic applications in several medical fields. Previous studies have indicated the activity of linalool against cell lines; however, its high level of toxicity restricts its use. The aim of this study was to design and manufacture compounds with a novel structure that can be used for loading linalool, to reduce its toxicity and improve its reachable ability. METHODS: We synthesized and characterized a new molecule for loading linalool onto gold nanoparticles (GNPs) capped with glutathione and conjugated with a CALNN peptide. Linalool was loaded onto the GNPs via the reaction of the surface groups of both linalool and the GNPs. Moreover, the target peptide could be loaded onto the surface of the GNPs via a chemical reaction. The cytotoxic effects of linalool-GNP (LG) and linalool-GNP-CALNN peptide (LGC) conjugates against ovarian cancer cells were investigated, as were the possible mechanisms underlying the induction of apoptosis. RESULTS: Our findings illustrated the significant antiproliferative effect of LG and LGC on SKOV-3 cells. The cytotoxicity assay demonstrated that LG and LGC were selectively toxic in cancer cells and induced apoptosis by activating caspase-8, the p53 protein, and various proteins involved in apoptosis. The present data demonstrated that LG and LGC have a high therapeutic potential and should be given particular consideration as anticancer drug-delivery systems, as LG and LGC were remarkably more cytotoxic against a cancer cell line than were linalool and GNPs alone. CONCLUSION: We concluded that LG and LGC are promising compounds that can be used for treating ovarian cancer (SKOV-3) cells via the induction of apoptosis through extrinsic and intrinsic pathways.

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness.

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.

Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats using the Thymidine Analog BrdU.

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2'-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.